Development of genomic resources in the domestic cat is key to application of this species as a model for heritable and infectious diseases, and for comparative genome analysis. Previous versions of the cat gene map, based on somatic cell hybrid analysis, revealed considerable conservation of synteny with the human genome. However, these maps provided no knowledge of gene order and genome rearrangement between the two species, information which is critical to applying the comparative candidate approach to positional cloning in gene-poor model systems. Radiation hybrid (RH) mapping has emerged as the most powerful tool for constructing moderate- to high-density gene maps in vertebrates by obviating the need to identify interspecific polymorphisms. A domestic cat RH panel has been developed and serves as the backbone for feline gene mapping, while also serving to cross-reference microsatellite positions on the current domestic cat-Asian leopard cat interspecies linkage map. We have completed a second generation 1150-locus map integrating 550 coding loci (expressed sequence tags, genes, and comparative anchor tagged sequences [CATS] markers) with 600 microsatellites selected from the current linkage map. The observed 100 conserved homologous gene segments between cat and human agree well with theoretical expectations (using the methods of Nadeau & Taylor), suggesting we have accurate cross-reference to the majority of the human genome. By comparison to the genetic map, the 1150-marker RH map has a mean density of 1 genetic marker/5 cM, now useful for comparative candidate positional clone analysis of the over 200 described feline hereditary phenotypes. We are currently increasing the type I marker density to nearly 800 loci, focusing primarily on gaps in human-cat synteny. Presently we are also increasing microsatellite coverage at least five-fold with an ultimate goal of 1000-1500 microsatellites on the map. Further refinement of rearrangement breakpoints and conserved segment boundaries will enhance cross-reference to other mammalian species' genomes for use in diverse studies of genetics and genome evolution.